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Analysis of Multipesticide Residuesin TobaccoThe Agilent Triple Quadrupole GC/MS/MS Analyzer forPesticides in Tobacco

Authors

Vivian Xianyu Chen and Andy Zhai

Agilent Technologies, Inc.

412 Yinglun Road

Shanghai 200131

China

Application Note

Food Testing & Agriculture

Abstract

A multiple reaction monitoring (MRM) method was developed and evaluated on an

Agilent 7000C Triple Quadrupole GC/MS for the analysis of 162 multiclass

pesticides in tobacco. This system was equipped with a Multimode Inlet, and

injections of 1 µL were made in the splitless mode. The midcolumn backflushing

technique was applied for time-effective elimination of less volatile matrix

components from the GC column. This reduced the cycle time and prevented

contamination of the MS ion source. A modified QuEChERS sample preparation

technique was used. Numerous tobacco samples were analyzed for method

validation. Key performance parameters investigated were linearity, recovery, %RSD,

limit of detection (LOD), and limit of quantitation (LOQ).

Assuming the water content in all tobacco samples was zero, this study showed

excellent recoveries (70 to 120%) for 95% of all test pesticides at 0.05 and

0.5 mg/kg (pesticide:tobacco, w:w), and LOQs of 0.01 mg/kg or lower for most

pesticides. We obtained excellent linearity from 0.01 to 2 mg/kg (pesticide:tobacco,

w:w), and repeatability over seven injections at concentration levels near LOQ, and

at 0.1 mg/kg in a tobacco matrix.

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Introduction

Tobacco, one of the world’s leading high-value crops, is proneto pest attack, so farmers apply various pesticides as controlmeasures. Pesticide residues on tobacco during cultivationcan remain in the leaves at harvest and even survivepostharvest processing treatments, eventually appearing inthe final products. Concerns regarding threats to humanhealth from the use of pesticides have prompted the tobaccoindustry worldwide to put more emphasis on the risks ofpesticide residues in tobacco. To protect consumers andcontrol pesticide residue levels, Guidance Residue Levels(GRLs) for 118 pesticides have been issued [1].

Multipesticide residue analysis in tobacco is a challenge inboth sample preparation and analytical detection. Tandemmass spectrometry (MS/MS) on a triple quadrupole platformis very useful for screening, confirming, and quantitatingtrace-level target compounds in these complex matricesbecause it can minimize interferences. GC/MS/MStechniques provide much better sensitivity and, thus,significantly lower system detection limits. For targetpesticide analysis in tobacco matrices, the Agilent 7000CTriple Quadrupole GC/MS/MS Analyzer for Pesticides inTobacco comes with a turnkey method for 162 pesticides. Italso includes an Agilent Pesticides and EnvironmentalPollutants MRM database (p/n G9250AA) of over 1,000compounds, which makes the analytical task easy andproductive.

The QuEChERS sample preparation technique [2,3,4] has beenrapidly accepted worldwide for multipesticide residue analysisdue to its attractive features, referred to as Quick, Easy,Cheap, Effective, Rugged, and Safe. QuEChERS extracts canbe analyzed by GC combined with MS to determine a widerange of pesticide residues. Agilent Bond Elut QuEChERSExtraction Kits have demonstrated excellent recoveries forfrequently used pesticides in different matrices [5]. However,tobacco extracts processed by QuEChERS are still verycomplex, containing various matrix residues such ashigh-boiling indigenous compounds.

QuEChERS extracts used in GC/MS/MS analysis can causecontamination and deterioration of the GC analytical columnand MS ion source. This results in poor data quality due topoor peak shape, retention time shifting, and loss ofresponses for active analytes. These extracts also lead toshorter lifetime of GC analytical columns and frequent MSmaintenance. To achieve low quantitation limits for pesticideanalysis, it is necessary to use the best techniques andsupplies to achieve reliable results, and to protect theanalytical column and MS ion source.

Backflushing the GC column ensures that high-boilingcompounds in the matrix do not pass through it, reducingcolumn bleed, eliminating ghost peaks, and minimizingcontamination of the mass spectrometer. Therefore, columnbackflushing can be beneficial for the analysis of tobaccoextracts because it significantly reduces analysis time, andreduces both column head trimming and the frequency of MSion source cleaning. Agilent capillary flow technology (CFT)makes column backflushing routine [6].

The Agilent Ultra Inert deactivation process significantlyimproves the inertness and robustness of wool liners. Thewool surface is deactivated thoroughly. Ultra Inert splitlessliners with wool have demonstrated excellent inertness inquantitative analysis of active and difficult pesticides in manymatrices. Ultra Inert liners with wool also protect the sampleflow path better, resulting in extended column lifetime andless frequent MS source maintenance [7].

This application note describes a study using midcolumnbackflushing and the 7000C Triple Quadrupole GC/MS tomeasure 162 pesticide residues in tobacco. The pesticideswere selected because of their presence in the CORESTA GRLlist [1], and in the list of pesticides banned or recommendedfor tobacco cultivation in China.

Experimental

Tobacco matrix blanks, extracted using the QuEChERSmethod, were spiked with pesticide working solutions. Thematrix-matched working calibration standards were thenanalyzed by GC/MS/MS using multiple reaction monitoring(MRM). A calibration curve from 0.01 to 2 mg/kg was used toevaluate linearity. Pesticide-free tobacco samples spiked withknown concentrations of pesticide solutions were extractedby QuEChERS for recovery, repeatability, limit of detection(LOD), and limit of quantitation (LOQ) studies. In all studies,the unit of mg/kg was based on pesticide:tobacco (w:w),assuming the water content in the tobacco samples was zero.

Chemicals and reagentsAll reagents and solvents were HPLC or analytical grade.Acetonitrile (ACN) was from J&K Scientific (Beijing, China).Toluene was from ANPEL Scientific Instrument (Shanghai,China). Water was from J. T. Baker. A set of pesticide stocksolutions in acetonitrile (100 µg/mL) and the internalstandard stock solution in acetonitrile (triphenyl phosphate,TPP), 1,000 µg/mL) were purchased from Ultra Scientific(North Kingstown, RI, USA).

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Reagent solution preparationThe pesticide composite intermediate solution (2 µg/mL) wasprepared by mixing and diluting the pesticide stock solutionswith acetonitrile:toluene (2:1, v:v). The Internal Standard(ISTD) intermediate solution (20 µg/mL) was prepared bydiluting the internal standard stock solution withacetonitrile:toluene (2:1, v/v).

Pesticide working solutions were used for preparation ofmatrix-matched standards. The working solutions containedISTD at a constant concentration and pesticide analytes atappropriate concentration levels. Pesticide compositeintermediate solution (2 µg/mL), ISTD intermediate solution(20 µg/mL), and acetonitrile:toluene (2:1, v:v) were used toprepare the working solutions.

Matrix-matched working calibration standards were derivedfrom blank tobacco extracts as described in the samplepreparation procedure. Appropriate pesticide workingsolutions were added to achieve matrix-matched workingcalibration standards from 0.01 to 2 mg/kg (pesticide:tobacco,w:w), assuming the water content in blank tobacco sampleswas zero.

Sample preparationPreparation of tobacco extracts was based on the modifiedEN version of the QuEChERS method. Two grams of tobaccowere extracted with 10 mL acetonitrile, diluted with 5 mLtoluene, extracted with an Agilent Bond Elut QuEChERSExtraction Kit (p/n 5982-5650), and cleaned up using anAgilent Bond Elut QuEChERS Dispersive Kit (p/n 5982-5022).ISTD was added to the sample before extraction to control theentire analytical process. The QuEChERS procedure is shownbelow.

Blank tobacco extracts were prepared in the same way as thesample treatment procedure, except there was no addition ofthe ISTD solution, and 1 mL final supernatant was blown tonear dryness under a nitrogen stream.

Weigh 2 g tobacco sample into a 50 mL tube

Add 10 mL water, mix uniformly, and let stand for 10 minutes

Add 10 mL ACN and ISDTs, vortex 2 minutes at 2,000 rpm

Place the tube into a refrigerator at –18 °C for 10 minutes

Add the salts, shake and vortex 2 minutes at 2,000 rpm

Centrifuge at 4,000 rpm for 5 minutes

Transfer 1.5 mL of supernatant to the dispersive SPE tube

Vortex 2 minutes at 2,000 rpm

Centrifuge at 4,000 rpm for 5 minutes

Transfer the supernatant to a sample vial for analysis

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InstrumentationThis study was performed on an Agilent 7890A GC coupled toan Agilent 7000C Triple Quadrupole GC/MS with an electronionization (EI) source. The GC system was equipped with anElectronic Pneumatics Control (EPC), a Multimode Inlet(MMI), an Agilent 7693A Automatic Liquid Sampler, and abackflushing system based on a Purged Ultimate Unioncontrolled by an AUX EPC module. Two Agilent J&W DB-5msUltra Inert GC 15 m columns were used to provide analyteseparation and a highly inert flow path into the detector.Agilent MassHunter Software was used for instrumentcontrol and for qualitative and quantitative data analysis.Retention time locking eliminated the need to adjust timesegment windows of MRM groups [8]. The run time was40.5 minutes with an extra 5 minutes for backflushing.

The Agilent MRM Database was used to help build the MSacquisition method for the target analytes by selecting theMRM transitions with minimum matrix interferences andmaximum responses. For each pesticide, two MRMtransitions were selected for quantitation and qualification,and the collision energy was optimized. However, differenttransitions might be used for quantitation in different tobaccomatrices to minimize matrix effects. Therefore, it is critical toreview the data in matrix before setting up a quantitationmethod.

Table 1 lists the instrument parameters used in this study.Table 2 shows the consumable supplies, and Table 3 thepesticides in alphabetical order with their quant and qualtransitions, and the collision energies for each.

Table 1. Instrumentation and analytical conditions.

GC conditions

Column 1 Agilent J&W DB-5ms UI, 15 m × 0.25 mm, 0.25 µm (p/n 122-5512UI),configured from MMI to AUX EPC

Column 2 Agilent J&W DB-5ms UI, 15 m × 0.25 mm, 0.25 µm (p/n 122-5512UI),configured from AUX EPC to vacuum

Carrier gas Helium

Injection mode Splitless

Injection volume 1 µL

Solvent washes Pre-injection2 × solvent A, acetonitrile, max volume2 × solvent B, toluene, max volumePost-injection5 × solvent A, acetonitrile, max volume5 × solvent B, toluene, max volume

Sample wash 1 × 3 µL

Sample pumps 3

Injection speed Fast

MMI temperature program 70 °C for 0 min, then 240 °C/min to 280 °C untilthe end of the analysis

Purge flow to split vent 50 mL/min at 1.25 min

Gas saver On 20 mL/min after 10 min

Septum purge flow 3 mL/min

Oven temperature program 60 °C for 1 min, then 40 °C/min to 120 °C, then 5 °C/min to 310 °C

Column 1 flow 1 mL/min

Column 2 flow 1.2 mL/min

Retention time locking Chlorpyrifos-methyl locked at 18.700 min

Run time 40.5 min

Post run 5 min at 310 °C, AUX EPC pressure 50 psi, inletpressure 2 psi

MS conditions

MS source EI, –70 eV

Source temperature 300 °C

Quadrupole temperature 180 °C

Transfer line temperature 280 °C

Solvent delay 3.75 min

Helium quench gas 2.25 mL/min

Nitrogen collision gas 1.5 mL/min

Acquisition mode Multiple reaction monitoring

MS1/MS2 resolution Wide

Time segments Refer to Table 3

Acquisition parameters Refer to Table 3

Table 2. Consumable supplies.

Vials Amber, write-on spot, 100/pk (p/n 5182-0716)

Vial caps Blue, screw cap, 100/pk (p/n 5182-0717)

Vial inserts 150 µL glass with polymer feet, 100/pk (p/n 5183-2088)

Septa Advanced green, 50/pk (p/n 5183-4759)

Inlet liners 4 mm id liner, UI, splitless, single taper, glass wool (p/n 5190-2293)

Agilent Bond Elut p/n 5982-5650QuEChERS Extraction Kits EN

Agilent Bond Elut p/n 5982-5022QuEChERS Dispersive Kits

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Table 3. Quantifier and qualifier MRM transitions for target pesticides*.

Quantifier Qualifier

Compound Transition CE Transition CE

3-Hydroxycarbofuran 180.0 & 137.0 15 137.0 & 107.0 15

Acephate 136.0 & 42.0 5 142.0 & 96.0 5

Acetamiprid 152.0 & 116.1 15 126.0 & 73.0 25

Acibenzolar-S-methyl 135.0 & 63.1 20 182.0 & 181.1 5

Alachlor 237.0 & 160.1 5 269.0 & 188.0 5

Aldrin 262.9 & 192.9 35 262.9 & 190.9 35

alpha-Endosulfan 262.8 & 192.9 30 195.0 & 160.0 10

Atrazine 214.9 & 58.1 10 214.9 & 200.2 5

Azinphos-ethyl 160.0 & 77.1 20 160.0 & 77.0 20

Azinphos-methyl 160.0 & 77.1 20 132.0 & 51.0 30

Azoxystrobin 344.1 & 171.9 40 344.1 & 329.0 15

Benalaxyl 266.0 & 148.1 5 233.9 & 146.0 20

Benfluralin 292.0 & 264.0 5 292.0 & 206.0 10

Benfuracarb 190.0 & 102.0 10 190.0 & 74.0 20

beta-Endosulfan 241.0 & 206.0 25 206.9 & 172.0 15

Bifenthrin 181.2 & 165.2 25 181.2 & 166.2 10

Bitertanol 170.1 & 141.1 20 170.1 & 115.0 40

Bromacil 205.0 & 188.0 15 207.0 & 190.0 15

Bromophos 330.8 & 315.8 15 328.8 & 313.8 15

Butralin 266.0 & 220.2 10 266.0 & 174.2 20

Cadusafos 158.8 & 97.0 15 157.9 & 96.9 15

Captafol 310.8 & 78.8 15 150.0 & 71.9 5

Captan 116.9 & 82.0 30 149.0 & 70.0 15

Carbaryl 144.0 & 115.1 20 144.0 & 116.1 10

Carbofuran 221.0 & 164.0 10 164.0 & 103.0 25

Carbosulfan 118.0 & 76.0 5 160.0 & 62.0 20

Chinomethionate 233.9 & 206.1 10 233.9 & 148.1 25(oxythioquinox)

Chlorantraniliprole 278.0 & 249.0 25 278.0 & 215.0 30

Chlordane (cis-) 372.9 & 265.9 20 271.9 & 236.9 15

Chlordane (trans-) 271.7 & 236.9 15 372.8 & 265.8 15

Chlordimeform 151.9 & 117.1 10 180.9 & 140.0 15

Chlorfenvinphos 266.9 & 159.1 15 268.9 & 161.0 15

Chlornitrofen 316.6 & 286.6 15 316.6 & 195.6 25

Chlorobenzilate 251.1 & 139.1 15 139.1 & 75.1 30

Chlorothalonil 263.8 & 133.0 40 264.0 & 168.0 25

Chlorpyrifos 196.9 & 169.0 15 198.9 & 171.0 15

Chlorpyrifos-methyl 285.9 & 92.9 20 124.9 & 47.0 15

Chlorthal-dimethyl (DCPA) 300.9 & 223.0 25 298.9 & 221.0 25

Clomazone 125.0 & 89.0 15 204.0 & 78.0 30

Cyfluthrin 226.0 & 206.0 15 162.9 & 127.0 5

Cyhalothrin (lambda) 208.0 & 181.0 5 181.1 & 152.0 25

Cypermethrin 165.1 & 127.1 5 163.1 & 127.1 5

Dazomet 161.9 & 89.0 5 89.0 & 46.0 15

DBCP 157.0 & 75.0 5 155.0 & 75.0 5

Deltamethrin 252.9 & 93.0 15 253.0 & 174.0 5

Demeton-O 88.0 & 60.0 5 171.0 & 115.0 10

Demeton-S 88.0 & 60.0 5 126.0 & 65.0 10

Demeton-S-methyl 88.0 & 60.0 5 142.0 & 78.9 10

Demeton-S-methyl sulfone 169.1 & 109.0 15 169.1 & 125.1 5

*Note: for analytes with multiple artifacts, the one with the highest response was analyzed and quantified.

Diazinon 304.0 & 179.0 15 137.1 & 84.0 10

Dichlorvos 184.9 & 93.0 10 185.0 & 109.0 15

Dicloran 160.1 & 124.1 10 206.1 & 176.0 10

Dieldrin 262.9 & 193.0 35 262.9 & 191.0 35

Difenoconazole 322.8 & 264.8 15 264.9 & 202.0 20

Diflubenzuron 153.0 & 125.0 15 153.0 & 91.0 30

Dimefox 110.0 & 47.0 35 153.0 & 110.0 10

Dimetachlone 187.0 & 152.0 5 243.0 & 187.0 10

Dimethoate 86.9 & 46.0 15 92.9 & 63.0 10

Dimethomorph (E) 301.0 & 139.0 15 300.9 & 165.0 10

Dimethomorph (Z) 301.0 & 139.0 15 300.9 & 165.0 10

Diphenamid 167.1 & 165.1 20 167.1 & 152.1 15

Disulfoton 88.0 & 60.0 5 142.0 & 81.0 10

Disulfoton sulfone 213.0 & 96.9 15 152.9 & 97.0 10

Disulfoton sulfoxide 97.0 & 65.0 20 212.0 & 97.0 30

Endosulfan-sulphate 271.9 & 237.0 15 273.8 & 238.9 15

Endrin 262.8 & 193.0 35 263.0 & 228.0 20

EPN 157.0 & 110.0 15 141.0 & 77.1 15

Ethion 230.9 & 129.0 20 231.0 & 175.0 10

Ethoprophos 157.9 & 97.0 15 157.9 & 114.0 5

Famoxadone 329.9 & 223.9 10 330.0 & 224.0 5

Fenamiphos (phenamiphos) 303.0 & 154.0 15 303.0 & 180.0 20

Fenamiphos sulfone 319.8 & 292.0 10 291.8 & 214.0 10

Fenamiphos-sulfoxide 304.0 & 196.0 5 304.0 & 122.0 15

Fenchlorphos (Ronnel) 285.0 & 269.9 15 286.9 & 272.0 15

Fenitrothion 277.0 & 260.1 5 277.0 & 109.0 15

Fensulfothion 291.8 & 156.0 15 292.8 & 96.8 20

Fenthion 278.0 & 169.0 15 278.0 & 109.0 15

Fenthion sulfone 309.9 & 105.0 10 124.9 & 47.0 10

Fenthion sulfoxide 125.0 & 47.0 10 278.0 & 109.0 15

Fenvalerate 224.9 & 119.0 15 167.0 & 125.1 5

Flucythrinate 156.9 & 107.1 15 198.9 & 107.0 25

Flumetralin 143.0 & 107.1 20 157.0 & 109.0 25

Folpet 260.0 & 130.0 15 261.8 & 130.1 15

Fonofos 245.9 & 137.0 5 245.9 & 109.0 15

Formothion 124.9 & 47.0 15 170.0 & 93.0 5

gamma-HCH (lindane) 219.0 & 183.0 5 181.0 & 145.0 15

HCH (alpha-) 216.9 & 181.0 5 219.0 & 183.0 5

HCH (beta-) 218.9 & 183.1 5 219.0 & 183.0 5

HCH (delta-) 217.0 & 181.1 5 219.0 & 183.1 5

Heptachlor 271.7 & 236.9 15 273.7 & 238.9 15

Heptachlor epoxides (cis-) 352.8 & 262.9 15 354.8 & 264.9 15

Heptachlor epoxides (trans-) 182.9 & 118.9 25 289.0 & 219.0 30

Heptenophos 124.0 & 89.0 10 124.0 & 63.0 35

Hexachlorobenzene 283.8 & 213.9 30 281.8 & 211.9 30

Indoxacarb 202.9 & 134.0 15 203.0 & 78.0 30

Iprobenfos 245.9 & 91.0 15 203.9 & 91.0 5

Iprodione 313.8 & 55.9 20 313.8 & 244.9 10

Isazophos 257.0 & 119.0 15 257.0 & 162.0 5

Isopropalin 264.0 & 222.2 5 238.0 & 165.2 10



Results and Discussion

RecoveryTable 4 shows the mean recoveries of three repetitive tobaccosamples at 0.05 and 0.5 mg/kg, respectively. For bothconcentration levels, 95% of the pesticides were in the rangeof 70 to 120%, showing excellent recoveries. The meanrecoveries of 70 to 120% and RSD ~ 20% represent widelyacceptable validation criteria in pesticide residue analysis, butother criteria could be used and justified depending on thepurpose of the analysis. For example, the Pesticide DataProgram (PDP) requires mean recoveries of 50 to 150% formethods used for analysis of PDP samples [9], because themain aim of PDP is to provide exposure data, and ideallyinclude as many pesticides as possible in multiresiduemethods.

If they are consistent (RSD ~ 20%), some compounds withrecoveries outside 70 to 120% can still be analyzed byQuEChERS, but may require special consideration.Compounds such as captan, chlorothalonil, diflubenzuron, andfolpet are base-sensitive and unstable even in acetonitrile.They tend to degrade in the presence of basic compounds (athigher pH), and often present issues in terms of recovery fromthe matrix and precision during analysis. Although not used inthis study, the evaluation of their corresponding internalstandards such as captan-d6 and folpet-d4 are recommended[10] to control recovery and ensure reliable results, especiallyfor longer batches in which the number of injections exceeds40. Compounds such as benfuracarb and carbosulfan areacid-sensitive and degrade at lower pH.

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Isoprothiolane 162.1 & 85.0 20 189.1 & 89.0 20

Leptophos 377.0 & 362.0 20 376.8 & 361.8 20

Malathion 172.9 & 99.0 15 126.9 & 99.0 5

Metalaxyl 234.0 & 146.1 20 234.0 & 174.1 10

Methamidophos 141.0 & 64.0 20 141.0 & 95.0 5

Methidathion 144.9 & 85.0 5 144.9 & 58.1 15

Methiocarb 168.0 & 109.1 15 168.0 & 91.0 30

Methiocarb sulfone 200.0 & 121.0 15 185.0 & 121.0 5

Methomyl 105.0 & 88.0 5 105.0 & 58.0 10

Methoprene 153.0 & 111.1 5 153.0 & 83.0 20

Methoxychlor 227.0 & 169.1 25 227.0 & 141.1 40

Metolachlor 238.0 & 162.2 10 162.2 & 133.2 15

Mevinphos 127.0 & 95.0 15 192.0 & 127.0 10

Mexacarbate 165.1 & 134.0 10 165.1 & 150.0 15

Mirex 271.8 & 236.8 15 273.8 & 238.8 15

Monocrotophos 192.0 & 127.1 10 127.1 & 95.0 15

Myclobutanil 179.0 & 125.1 10 150.0 & 123.0 15

Naled 144.9 & 109.0 15 184.9 & 93.0 15

Napropamide 271.0 & 72.1 15 128.0 & 72.1 5

Nitrofen 202.0 & 139.1 20 282.9 & 253.0 10

o,p'-DDD 235.0 & 165.2 20 237.0 & 165.2 20

o,p'-DDE 246.0 & 176.2 30 248.0 & 176.2 30

o,p'-DDT 235.0 & 165.0 20 237.0 & 165.0 20

Omethoate 155.9 & 110.0 5 155.9 & 79.0 20

Oxadixyl 163.0 & 132.1 5 232.9 & 146.1 10

Oxamyl 98.0 & 58.0 10 145.0 & 71.9 20

p,p'-DDD 235.0 & 165.0 20 237.0 & 165.0 20

p,p'-DDE 315.8 & 246.0 15 246.1 & 176.2 30

p,p'-DDT 235.0 & 165.0 20 237.0 & 165.0 20

Parathion 290.9 & 109.0 10 138.9 & 109.0 5

Parathion-methyl 262.9 & 109.0 10 232.9 & 109.0 10

Penconazole 248.0 & 192.1 15 248.0 & 157.1 25

Pendimethalin (penoxaline) 251.8 & 162.2 10 251.8 & 161.1 15

Permethrin 163.0 & 127.0 5 183.1 & 165.1 10

Phorate 121.0 & 47.0 30 260.0 & 75.0 10

Phosalone 182.0 & 75.1 30 182.0 & 111.0 15

Phosphamidon (E) 264.0 & 127.0 15 264.0 & 72.0 10

Phosphamidon (Z) 264.0 & 127.0 15 192.9 & 127.0 5

Piperonyl butoxide 176.1 & 103.1 25 176.1 & 131.1 15

Pirimicarb 238.0 & 166.2 10 166.0 & 55.1 20

Pirimiphos-methyl 290.0 & 125.0 20 232.9 & 151.0 5

Profenofos 338.8 & 268.7 15 207.9 & 63.0 30

Propoxur 110.0 & 63.0 25 110.0 & 64.0 15

Prothiofos 308.9 & 238.9 15 266.9 & 239.0 5

Pyrazophos 221.0 & 193.1 10 232.0 & 204.1 10

Quinalphos 298.0 & 156.0 20 157.0 & 102.0 30

Quizalofop-p-ethyl 371.8 & 298.9 10 163.0 & 136.0 10

Schradan 153.1 & 46.1 15 199.0 & 92.0 5

Tefluthrin 177.1 & 87.0 30 177.1 & 127.1 15

Teflubenzuron 197.0 & 135.0 30 197.0 & 142.0 25

Terbufos 230.9 & 129.0 20 230.9 & 175.0 10

Terbufos sulfone 264.0 & 97.0 25 199.0 & 97.0 20

Tetrachlorvinphos 330.8 & 108.9 15 328.8 & 108.9 15

Tetradifon 158.9 & 111.0 20 353.8 & 226.8 10

Thiamethoxam 212.0 & 139.0 15 212.0 & 125.0 10

Thionazin 143.0 & 79.0 10 175.0 & 79.0 10

Triadimefon 208.0 & 181.1 5 208.0 & 111.0 20

Triadimenol 168.0 & 70.0 10 128.0 & 65.0 25

Triazophos 257.0 & 162.1 5 161.2 & 134.2 5

Trichlorfon 145.0 & 109.0 12 109.0 & 79.0 10

Triflumuron 139.0 & 75.0 30 139.0 & 111.0 15

Trifluralin 305.9 & 264.0 5 290.0 & 248.0 5

Uniconazole 234.1 & 137.0 15 234.1 & 165.1 10

Vamidothion 141.9 & 78.9 10 145.0 & 87.0 5

Table 3. Quantifier and qualifier MRM transitions for target pesticides*.Quantifier Qualifier




Note: for analytes with multiple artifacts, the one with the highest response was analyzed and quantified.

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Table 4. Method validation data: linearity, recovery (n = 3), %RSD (n = 3), LOD, and LOQ of 162 pesticides.

3-Hydroxycarbofuran 0.01-2 0.9958 95.26 11.98 106.21 6.17 0.0416 0.1388

Acephate 0.01-2 0.9973 78.95 9.89 76.12 4.11 0.0036 0.0121

Acetamiprid 0.01-2 0.9985 101.3 2.34 107.03 2.11 0.0074 0.0246

Acibenzolar-S-methyl 0.01-2 0.9976 88.61 0.48 98.16 1.65 0.0014 0.0045

Alachlor 0.01-2 0.9984 99.29 3.2 100.68 1.86 0.0015 0.0052

Aldrin 0.01-2 0.9997 92.71 0.93 105.2 0.86 0.002 0.0068

alpha-Endosulfan 0.01-2 0.9993 101.24 4.88 108.07 2.46 0.0026 0.0088

Azinphos-ethyl 0.01-2 0.9925 92.8 2.32 94.73 2.32 0.0018 0.0059

Azinphos-methyl 0.01-2 0.9942 103.94 5.11 88.6 0.57 0.0213 0.0711

Azoxystrobin 0.01-2 0.9977 104.3 6.05 108.05 1.68 0.0037 0.0125

Benalaxyl 0.01-2 0.9996 104.12 2.07 106.13 2.77 0.001 0.0034

Benfluralin 0.01-2 0.9796 86.6 3.81 85.13 2.57 0.0023 0.0077

Benfuracarb 0.01-2 0.9990 78.45 2.82 78.57 3.09 0.0023 0.0077

beta-Endosulfan 0.01-2 0.9993 103.62 5.35 105.88 1.83 0.0067 0.0223

Bifenthrin 0.01-2 0.9999 103.37 1.28 108.8 1.61 0.0026 0.0088

Bitertanol 0.01-2 0.9994 103.58 2.89 106.16 1.45 0.0027 0.0089

Bromacil 0.01-2 0.9992 96.42 1.63 101.98 1.86 0.0013 0.0044

Bromophos 0.01-2 0.9990 95.63 1.96 102.49 1.6 0.0012 0.0041

Butralin 0.01-2 0.9764 86.01 2.22 84.08 2.05 0.0025 0.0083

Cadusafos 0.01-2 0.9990 94.92 5.19 100.05 2.46 0.0026 0.0086

Captafol 0.01-2 0.9991 94.37 22.53 74.16 10.38 0.0222 0.0739

Captan 0.01-2 0.9954 77.23 3.74 79.66 9.92 0.0061 0.0204

Carbaryl 0.01-2 0.9991 98.84 0.78 102.09 2.79 0.0032 0.0107

Carbofuran 0.01-2 0.9967 113.25 13.81 107.89 1.65 0.0134 0.0446

Carbosulfan 0.01-2 0.9980 83.01 5.14 98.27 0.6 0.0018 0.0059

Chinomethionate (oxythioquinox) 0.01-2 0.9989 63.97 5.88 58.37 2.49 0.0017 0.0056

Chlorantraniliprole 0.01-2 0.9982 129.44 2.96 123.13 3.23 0.004 0.0132

Chlordane (cis-) 0.01-2 0.9994 94.74 7.3 105.68 2.6 0.0031 0.0103

Chlordane (trans-) 0.01-2 0.9995 97.11 2.41 105.74 1.86 0.0027 0.0091

Chlordimeform 0.01-2 0.9996 92.57 2.85 93.24 1.97 0.0026 0.0086

Chlorfenvinphos 0.01-2 0.9989 101.32 3.62 104.28 1.26 0.0011 0.0037

Chlornitrofen 0.01-2 0.9886 92.29 1.41 93.14 3.44 0.0039 0.0129

Chlorobenzilate 0.01-2 0.9996 101.61 1.95 107.34 1.65 0.0011 0.0037

Chlorothalonil 0.01-2 0.9958 31.84 15.52 46.19 3.11 0.0019 0.0065

Chlorpyrifos 0.01-2 0.9992 95.06 6.73 105.14 1.95 0.0025 0.0085

Chlorpyrifos-methyl 0.01-2 0.9982 94.51 3.18 100.01 1.85 0.0023 0.0076

Chlorthal-dimethyl (DCPA) 0.01-2 0.9996 99.72 3.62 106.3 1.58 0.0008 0.0027

Clomazone 0.01-2 0.9995 97.78 2.71 100.83 3.97 0.0014 0.0045

Cyfluthrin 0.01-2 0.9940 99.34 4.3 99.79 2.14 0.0014 0.0047

Cyhalothrin (lambda) 0.01-2 0.9931 96.48 3.61 95.36 0.57 0.0012 0.004

Cypermethrin 0.01-2 0.9927 103.83 5.85 101.93 1.04 0.0025 0.0083

Dazomet 0.01-2 0.9971 78.53 7.59 88.67 2.76 0.003 0.0099

DBCP 0.01-2 0.9999 86.41 0.64 93.17 6.56 0.0019 0.0062

Deltamethrin 0.01-2 0.9864 95.48 6.67 91.64 2.23 0.003 0.0101

Demeton-O 0.01-2 0.9991 107.06 10.43 101.67 2.54 0.0017 0.0057

Demeton-S 0.01-2 0.9980 94.01 6.46 98.75 2.47 0.002 0.0065

Demeton-S-methyl 0.01-2 0.9979 93.46 6.69 99.2 2.36 0.004 0.0133

Demeton-S-methyl sulfone 0.01-2 0.9945 90.44 4.22 86.32 3.97 0.0031 0.0103

Diazinon 0.01-2 0.9992 95.75 3.04 100.53 3.04 0.003 0.0099

Dichlorvos 0.01-2 0.9999 112.03 5.74 122.71 3.44 0.0037 0.0122

Recovery and %RSD

Linearity 0.05 mg/kg 0.5 mg/kg

Pesticide Linear range(mg/kg) R2

Recovery (%) %RSD

Recovery (%) %RSD

LOD (mg/kg)

LOQ (mg/kg)

8

Dicloran 0.01-2 0.9950 93.75 2.11 95.61 4.68 0.0028 0.0092

Dieldrin 0.01-2 0.9998 105.82 5.78 108.27 2.73 0.0043 0.0142

Difenoconazole 0.01-2 0.9985 105.12 2.14 108.8 1.27 0.0034 0.0115

Dimefox 0.01-2 0.9997 90.95 8.55 94.64 5.17 0.0036 0.0121

Dimetachlone 0.01-2 0.9998 99.98 5.29 105.32 1.69 0.003 0.01

Dimethoate 0.01-2 0.9960 97.04 5.59 98.46 0.61 0.003 0.0101

Dimethomorph (E) 0.01-2 0.9990 104.55 4.02 109.43 1.11 0.0017 0.0055

Dimethomorph (Z) 0.01-2 0.9997 105.95 3.4 113.19 2.29 0.0028 0.0093

Diphenamid 0.01-2 0.9996 101.55 4.29 105.3 1.21 0.0011 0.0036

Disulfoton 0.01-2 0.9984 92.8 1.86 99.38 2.75 0.0023 0.0075

Disulfoton sulfone 0.01-2 0.9965 94.79 5.19 99.66 0.95 0.0026 0.0088

Disulfoton sulfoxide 0.01-2 0.9976 105.45 8.03 104.96 8.06 0.003 0.01

Endosulfan-sulphate 0.01-2 0.9995 102.74 2.37 106.35 1.13 0.0009 0.0031

Endrin 0.01-2 0.9994 94.83 3.33 105.86 1.11 0.0039 0.0129

EPN 0.01-2 0.9946 96.29 3.99 97.99 2.26 0.0014 0.0048

Ethion 0.01-2 0.9959 97.75 4.41 101.84 2.27 0.0016 0.0052

Ethoprophos 0.01-2 0.9989 93.44 5.69 100.76 1.57 0.0033 0.0108

Famoxadone 0.01-2 0.9845 96.3 7.56 94.2 1.15 0.0039 0.0131

Fenamiphos (phenamiphos) 0.01-2 0.9991 99.23 3.11 102.83 1.86 0.0023 0.0075

Fenamiphos sulfone 0.01-2 0.9979 101.7 3.25 102.54 1.47 0.0013 0.0045

Fenamiphos-sulfoxide 0.01-2 0.9840 97.96 6.87 86.91 0.81 0.002 0.0067

Fenchlorphos (Ronnel) 0.01-2 0.9990 97.07 4.74 104.12 0.96 0.002 0.0065

Fenitrothion 0.01-2 0.9804 86.01 3.82 86.01 0.74 0.0026 0.0085

Fensulfothion 0.01-2 0.9874 113.76 2.77 120.81 3.12 0.0028 0.0094

Fenthion 0.01-2 0.9994 97.7 5.98 103.19 0.1 0.0024 0.0078

Fenthion sulfone 0.01-2 0.9979 93.67 6.96 97.65 2.82 0.0031 0.0102

Fenthion sulfoxide 0.01-2 0.9976 95.04 4.15 96.24 1.56 0.002 0.0066

Fenvalerate 0.01-2 0.9878 112.17 1.57 95.4 2.55 0.0072 0.0241

Flucythrinate 0.01-2 0.9959 106.16 2.37 106.38 2.89 0.0009 0.0031

Flumetralin 0.01-2 0.9904 90.29 5.57 93.81 1.97 0.0023 0.0076

Folpet 0.01-2 0.9906 75.72 5.77 71.03 8.01 0.0025 0.0082

Fonofos 0.01-2 0.9985 93.59 3.44 98.78 2.8 0.0017 0.0056

Formothion 0.01-2 0.9971 86.49 2.55 87.42 3.52 0.0029 0.0098

gamma-HCH (lindane) 0.01-2 0.9988 93.99 1.46 96.48 4.32 0.0013 0.0043

HCH (alpha-) 0.01-2 0.9991 94.48 2.64 100.32 3.89 0.0015 0.0049

HCH (beta-) 0.01-2 0.9981 97.69 3.91 98.73 3.95 0.0012 0.0039

HCH (delta-) 0.01-2 0.9990 96.07 3.67 97.4 4.18 0.0013 0.0043

Heptachlor 0.01-2 0.9967 89.21 3.27 98.28 2.77 0.0013 0.0043

Heptachlor epoxides (cis-) 0.01-2 0.9993 95.93 3.73 105.75 2.06 0.002 0.0066

Heptachlor epoxides (trans-) 0.01-2 0.9996 104.44 6.65 106.73 2.91 0.0037 0.0125

Heptenophos 0.01-2 0.9989 93.56 5.38 99.32 2.22 0.0029 0.0098

Hexachlorobenzene 0.01-2 0.9997 83.24 2.29 95.74 3.13 0.0015 0.0051

Indoxacarb 0.01-2 0.9998 106.45 3.22 115.42 1.11 0.003 0.0099

Iprobenfos 0.01-2 0.9968 96.43 3.84 100.83 1.52 0.0035 0.0117

Iprodione 0.01-2 0.9999 99.45 5.51 105.61 2.37 0.0025 0.0085

Isazophos 0.01-2 0.9988 97.65 2.42 98.61 5.92 0.0029 0.0096

Isopropalin 0.01-2 0.9822 87.37 1.75 87.92 1.55 0.0024 0.0081

Isoprothiolane 0.01-2 0.9996 101.5 1.29 106.6 1.06 0.0009 0.0029

Leptophos 0.01-2 0.9991 96.11 3.87 104 1.77 0.0016 0.0052

Malathion 0.01-2 0.9976 96.68 4.77 100.62 1.21 0.0021 0.007

Metalaxyl 0.01-2 0.9997 99.25 4.16 105.66 1.52 0.0026 0.0087

Recovery and %RSD



Recovery (%) %RSD

Recovery (%) %RSD

LOD (mg/kg)

LOQ (mg/kg)

9

Methamidophos 0.01-2 0.9985 64.8 9.05 72.27 3.05 0.004 0.0134

Methidathion 0.01-2 0.9980 94.26 2.82 98.29 1.07 0.0023 0.0075

Methiocarb 0.01-2 0.9993 97.78 4.25 102.84 1.89 0.0017 0.0057

Methiocarb sulfone 0.01-2 0.9973 92.5 2.96 100.65 1.07 0.0035 0.0118

Methomyl 0.01-2 0.9970 120.17 10.64 106.37 10.74 0.006 0.0201

Methoprene 0.01-2 0.9988 107.7 10.08 104.43 3.23 0.0179 0.0595

Methoxychlor 0.01-2 0.9970 93.03 1.39 94.25 5.39 0.0022 0.0072

Metolachlor 0.01-2 0.9991 97.92 2.1 103.27 1.29 0.0009 0.003

Mevinphos 0.01-2 0.9992 95.16 4.22 97.9 1.37 0.0058 0.0195

Mexacarbate 0.01-2 0.9991 99.65 4.42 101.16 3.76 0.0021 0.007

Mirex 0.01-2 0.9995 88.83 1.75 103.68 1.92 0.001 0.0032

Monocrotophos 0.01-2 0.9946 88 10.24 90.02 2.7 0.0052 0.0172

Myclobutanil 0.01-2 0.9997 103.08 1 106.29 1.24 0.0013 0.0044

Naled 0.01-2 0.9872 27.35 2.48 15.82 18.06 0.0019 0.0062

Napropamide 0.01-2 0.9995 101.99 1.08 105.75 1.08 0.0025 0.0083

Nitrofen 0.01-2 0.9924 93.4 2.75 94.42 1.19 0.0028 0.0095

o,p'-DDD 0.01-2 0.9997 101.75 0.8 110.48 1.03 0.001 0.0034

o,p'-DDE 0.01-2 0.9997 95.92 1.07 107.07 0.81 0.0015 0.0051

o,p'-DDT 0.01-2 0.9975 95.75 1.18 100 4.94 0.0021 0.007

Omethoate 0.01-2 0.9961 83.31 11.12 82.39 4.61 0.0037 0.0123

Oxadixyl 0.01-2 0.9995 106.24 3.18 106.29 1.77 0.0015 0.0051

Oxamyl 0.01-2 0.9971 107.18 8.43 96.25 5.86 0.0207 0.0689

p,p’-DDD 0.01-2 0.9991 105.79 2.1 109.09 1.4 0.001 0.0032

p,p’-DDE 0.01-2 0.9990 158.52 6.65 111.94 2.2 0.0105 0.035

p,p’-DDT 0.01-2 0.9967 100.53 3.45 95.5 6.79 0.0036 0.0119

Parathion 0.01-2 0.9754 88.29 3.98 86.33 1.52 0.003 0.0099

Parathion-methyl 0.01-2 0.9863 87.19 4.1 88.47 3.18 0.0028 0.0094

Penconazole 0.01-2 0.9994 100.09 3.94 103.44 0.47 0.0018 0.0059

Pendimethalin (penoxaline) 0.01-2 0.9769 82.87 3.16 83.8 1.8 0.003 0.0099

Permethrin 0.01-2 0.9993 102.01 0.87 107.26 1.24 0.002 0.0066

Phorate 0.01-2 0.9978 94.43 3.97 98.14 2.82 0.0029 0.0095

Phosalone 0.01-2 0.9986 96.62 2.4 100.12 1.4 0.0012 0.0039

Phosphamidon (E) 0.01-2 0.9971 93.1 13.39 98.58 3.89 0.0012 0.0041

Phosphamidon (Z) 0.01-2 0.9972 97.94 4.64 98.44 2.45 0.0015 0.005

Piperonyl butoxide 0.01-2 0.9996 110.28 1.59 106.4 2.06 0.0023 0.0075

Pirimicarb 0.01-2 0.9997 101.58 1.97 103.44 1.41 0.0014 0.0047

Pirimiphos-methyl 0.01-2 0.9981 95.16 4.76 101.25 1.21 0.0017 0.0058

Profenofos 0.01-2 0.9988 98.56 2.31 104.9 1.83 0.0024 0.0082

Propoxur 0.01-2 0.9993 97.23 4.97 103.62 1.99 0.0026 0.0087

Prothiofos 0.01-2 0.9989 97.98 1.05 103.32 1.17 0.0016 0.0052

Pyrazophos 0.01-2 0.9970 99.63 1.98 100.77 1.4 0.0015 0.005

Quinalphos 0.01-2 0.9984 99.95 3.3 101.18 3.7 0.0041 0.0138

Quizalofop-p-ethyl 0.01-2 0.9988 102.46 4.23 107.96 1.99 0.0014 0.0048

Schradan 0.01-2 0.9939 94.61 7.7 85.95 3.14 0.0023 0.0076

Tefluthrin 0.01-2 0.9996 101.33 1.27 105.38 3.87 0.0014 0.0048

Teflubenzuron 0.01-2 0.9996 95.89 6.64 99.87 5.37 0.0016 0.0052

Terbufos 0.01-2 0.9965 92.01 5.13 97.03 3.05 0.0024 0.0079

Terbufos sulfone 0.01-2 0.9972 91.68 4.51 100.41 2.53 0.0013 0.0042

Tetrachlorvinphos 0.01-2 0.9989 97.53 1.72 99.85 1.16 0.0025 0.0082

Tetradifon 0.01-2 0.9997 103.63 2.25 106.68 2.12 0.0008 0.0028

Thiamethoxam 0.01-2 0.9986 101.04 3.29 100.03 1.66 0.0018 0.006

Recovery and %RSD



Recovery (%) %RSD

Recovery (%) %RSD

LOD (mg/kg)

LOQ (mg/kg)

10

Thionazin 0.01-2 0.9988 94.57 5.34 100.92 2.39 0.0028 0.0094

Triadimefon 0.01-2 0.9994 97.77 3.36 103.18 2.13 0.002 0.0066

Triadimenol 0.01-2 0.9998 96.31 4.96 104.37 0.66 0.0017 0.0058

Triazophos 0.01-2 0.9976 101.33 1.78 101.32 2.38 0.0022 0.0073

Trichlorfon 0.01-2 0.9970 86.41 12 84.19 5.46 0.0042 0.0138

Triflumuron 0.01-2 0.9993 96.79 4.56 103.99 1.55 0.0031 0.0103

Trifluralin 0.01-2 0.9887 91.83 6.06 92.37 2.27 0.0034 0.0113

Uniconazole 0.01-2 0.9995 102.08 2.6 104.09 0.82 0.0025 0.0084

Vamidothion 0.01-2 0.9944 86.29 11.05 85.34 2.68 0.0024 0.0081

Recovery and %RSD



Recovery (%) %RSD

Recovery (%) %RSD

LOD (mg/kg)

LOQ (mg/kg)

Backflushing the columnThe GC/MS/MS system used a Purged Ultimate Union (PUU),and its configuration is shown in Figure 1. Backflushing wasdone for 5 minutes after the run by raising the pressure at thePUU and lowering the inlet pressure. This reversed the flowthrough the column and purged high-boiling matrixcomponents from the head of the column, out through theinlet's split vent. With the PUU installed, inlet and GC columnmaintenance is possible without venting the massspectrometer. Figure 2 shows that 5 minutes of backflushingcleaned the analytical columns and reduced the cycle time fortarget analytes in tobacco extracts.

During the course of this study, approximately 200+ 1 µLinjections of concentrated tobacco extracts were made intothe GC/MS/MS system with no evidence of column or MSperformance problems, as shown in Figure 3.

Pressure/Flowcontroller

Column 115 m × 0.25 mm, 0.25 µm

Agilent DB-5ms UI

Column 215 m × 0.25 mm, 0.25 µm

Agilent DB-5ms UI

Agilent 7000C or 7010 MS/MS

MMI1 µL Splitless injection

A

B

C

Agilent 7890 GC

RT locking compound – chlorpyrifos-methyl at 18.700 minutes

Total run time = 40.5 minutesPost run time(backflush) = 5 minutes

Figure 1. Hardware diagram of the Agilent Tobacco Analyzer.The GC/MS/MS system used for MRM analysis wasconfigured with A) Multimode Inlet, B) Purged UltimateUnion, and C) two Agilent J&W DB-5ms UI, 15 m × 0.25 mm,0.25 µm, GC columns.

11

Figure 3. Chromatograms of clomazone, triadimefon, myclobutanil, and permethrin at 0.01 mg/kg after 200+ 1 µL injectionsof concentrated tobacco extracts.

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 710

0.20.40.60.81.01.21.41.6×107

C

Acquisition time (min)

Counts

×108B

Counts

0.2

0.6

1.0

1.4

1.8

2.2 An additional 15+ minutes and heating the column to 310 °C was required to remove these high-boiling compounds

Run stopped at 40.5 minutes and backflush at 310 °C for 5 minutes

RT range of analytes

Blank run after backflushing showing the column was clean

0.2

0.6

1.0

1.4

1.8

2.2

×108A

Counts

Figure 2. Backflushing cleaned the analytical columns and reduced the cycle time for target analytes intobacco extracts.

+ MRM (125.0 & 89.0)

Acquisition time (min)16.0 16.2 16.4 16.6

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

Cou

nts

0

0.2

0.4

0.6

0.8

1

1.216.213 min


0

0.2

0.4

0.6

0.8

1

1.2

125.0 & 89.0, 204.0 & 78.0Ratio = 34.5 (28.4–42.6)

Clomazone+ MRM (208.0 & 181.1)


00.5

11.5

22.5

33.5

44.5

20.969 min


00.5

11.5

22.5

33.5

44.5

208.0 & 181.1, 208.0 & 111.0Ratio = 75.7 (59.1–88.7)

Triadimefon

+ MRM (179.0 & 125.1)

Acquisition time (min)24.3 24.5 24.7

00.10.20.30.40.50.60.70.80.9

24.577 min


00.10.20.30.40.50.60.70.80.9

179.0 & 125.1, 150.0 & 123.0Ratio = 22.6 (19.4–29.1)

Myclobutanil

+ MRM (163.0 & 127.0)


00.5

11.5

2

2.53

*32.660 min


0

12345

6

163.0 & 127.0, 183.1 & 165.1Ratio = 193.1 (88.9–133.3)

Permethrin

×104 ×104 ×103

×103

×103

×103

×104 ×104

12

Accurate calibrationA set of eight calibration matrix standards were injectedconsecutively, and yielded coefficient of correlation values(R2) that were > 0.99 in over 92% of the cases. Naled,trifluralin, benfluralin, parathion-methyl, fenitrothion,isopropalin, pendimethalin, fensulfothion, chlornitrofen,fenamiphos-sulfoxide, fenvalerate, deltamethrin, andfamoxadone yielded R2 values from 0.98 to 0.99. Parathionand butralin yielded R2 values from 0.97 to 0.98. Figure 4shows calibration curves obtained in the tobacco matrix forsix commonly detected pesticides in tobacco, namelytriadimefon, metalaxyl, isoprothiolane, clomazone,acetamiprid, and cyfluthrin.

Some pesticides, especially organophosphates, degrade insolvents. For these pesticides, pure solid phase standards aresuggested for storage. They need to be diluted and analyzedin a timely manner to achieve better linearity. The current MSgain factor setting in this study was 10. This may be loweredto achieve a larger linear range, if necessary.

LOQs well below MRLsThe LOD and LOQ calculations follow the EPA model, which isapproached with replicates using a t-value at 99% confidence[11]. In this study, seven repetitive injections of tobacco matrixblanks with known low concentration levels of spikedpesticides were analyzed to calculate LOD and LOQ. LOQs of all162 pesticides were well below the GRLs issued by ACAC [1].

Although all 162 pesticides can be analyzed with GC/MS, toachieve even lower LOQs, the preferred technique for somepesticides is LC/MS. These include some benzoylureainsecticides such as diflubenzuron, teflubenzuron, andtriflumuron; some carbamates such as benfuracarb, carbaryl,carbofuran, 3-hydroxycarbofuran, carbosulfan, methiocarb,methomyl, oxamyl, and pirimicarb; more polar or otherwiseproblematic organophosphates such as acephate, azinphos-Et/Me, dimethoate, methamidophos, monocrotophos, naled,and omethoate; some neonicotinoids such as acetamipiridand thiamethoxam; and nonhalogenated pyrethroids.

Triadimefon

y = 0.807738*x + 2.128478E–4

R2 = 0.9994

Metalaxyl Isoprothiolane

Clomazone Acetamiprid Cyfluthrin

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

00.20.40.60.81.01.21.41.6

×10–1 ×10–1

Rel

ativ

e re

spon

se

Relative concentration

y = 0.460464*x + 2.811171E–4

R2 = 0.9997

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

00.10.20.30.40.50.60.70.80.9

Rel

ativ

e re

spon

se


y = 1.117237*x + 6.481064E–4

R2 = 0.9996

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

00.20.40.60.81.01.21.41.61.82.02.2

Rel

ativ

e re

spon

se


y = 2.033208*x + 4.841767E–4

R2 = 0.9996

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

00.40.81.21.62.02.42.83.23.64.0

Rel

ativ

e re

spon

se


y = 0.301193*x – 4.983957E–4

R2 = 0.9985

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

0

1

2

3

4

5

6

0

1

2

3

4

5

6

Rel

ativ

e re

spon

se


y = 0.313208*x – 0.001383R2 = 0.9940

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Rel

ativ

e re

spon

se


Figure 4. Calibration curves in a tobacco matrix for representative pesticides triadimefon, metalaxyl, isoprothiolane,clomazone, acetamiprid, and cyfluthrin.

13

Other features that can affect LOQs include pesticide stabilityand the lack of characteristic precursor ions using electronionization. When using electron ionization, pesticides such ascaptafol and methoprene have fragmented EI spectra and lackcharacteristic precursors, product ions, or MRM transitions,that affect their LOQs. Degradation of pesticides is also acommon reason why some pesticides do not have good LOQs.Because naled and trichlorfon degrade to dichlorvos even atroom temperature, these three pesticides are sometimesanalyzed together and quantified using dichlorvos, only.Teflubenzuron degrades to three main artifacts, and only theartifact with the highest response was analyzed in this study.Captan not only has a fragmented EI spectrum, but also tendsto degrade to 1,2,3,6-tetrahydrophthalimide, which affect itsLOD and LOQ.

Excellent RSDsFigure 5A shows seven consecutive injections of each sampleat concentration levels near LOQ and 0.1 mg/kg, respectively.Of 162 pesticides tested, 159 were at the concentration levelnear LOQ, and 161 at 0.1 mg/kg yielded %RSDs less than15%.

Figure 5B shows the number of pesticides in tobacco withgiven %RSD values based on calculated amounts at twoconcentrations, 0.05 and 0.5 mg/kg. Percentage RSD valueswere obtained from three repetitive recovery study samples,and each was injected once on the GC/MS/MS. Of 162pesticides tested, 160 at 0.05 mg/kg (98.8%), and 161 at0.5 mg/kg (99.4%) yielded %RSD less than 15%.

In both studies, repeatability was excellent, showing lessthan 15% RSD for over 95% of the pesticides, even for themost challenging compounds omethoate, acephate, and DDT.Some pesticides, such as p,p’-DDT and methoxychlor, havesimilar structures and are known to degrade in the GC inlet,causing signal variability. In such cases, a suitable,compound-specific ISTD can be added to the final extractbefore instrumental analysis for signal normalization. Forp,p’-DDT, the postextraction addition of a labeled ISTD(13C12-p,p’-DDT) can be a cost-effective way to addressdegradation and other potential GC-related issues.

Figure 5. Distribution of %RSD of 162 pesticides in tobaccomatrices: (A) seven consecutive injections of one sample atthe concentration level near LOQ and at 0.1 mg/kg, (B) threerepetitive recovery study samples at two differentconcentration levels of 0.05 and 0.5 mg/kg.

0

10

20

30

40

50

60

70

80

90

100

0 ~ x ~ 5 5 < x ~ 10 10 < x ~ 15 15 < x ~ 20 20 < x ~ 100

Num

ber o

f pes

tici

des

%RSD range

0 ~ x ~ 5 5 < x ~ 10 10 < x ~ 15 15 < x ~ 20 20 < x ~ 100

%RSD range

0

20

40

60

80

100

120

140

160

Num

ber o

f pes

tici

des

A

B

~ LOQ

0.1 mg/kg

0.05 mg/kg0.5 mg/kg

14

Matrix effectsSome pesticides showed consistent responses in differentmatrices, but some pesticides had different responses indifferent matrices due to either matrix enhancement or matrixsuppression, and sometimes even slightly different retentiontime. Disulfoton sulfoxide, formothion, heptenophos, andtefluthrin spiked at 0.1 mg/kg were used as probes. Figure 6shows the matrix effect of three different tobacco matrices

A Matrix 1

BMatrix 2

C Matrix 3

D Matrix 1

E Matrix 2

F Matrix 3

Disulfoton sulfoxide Heptenophos TefluthrinFormothion

Figure 6. Chromatograms of disulfoton sulfoxide, formothion, heptenophos, and tefluthrin in three different tobacco matrices:(A-C) matrix blanks of three different tobacco matrices, (D-F) spiked at 0.1 mg/kg in three different matrix blanks.

from two different countries. The matrix enhancement ofdisulfoton sulfoxide and formothion in tobacco matrix 3 wasstronger than the one in tobacco matrices 1 and 2. Theretention time of disulfoton sulfoxide was also affected bydifferent matrices. Heptenophos and tefluthrin showedconsistent results in all three matrices. Therefore, matrixeffect was compound dependent and it is important to usematrix-matched calibrations to achieve accurate quantitationresults.

15

Conclusions

The Agilent 7000C Triple Quadrupole GC/MS/MS Analyzer forPesticides in Tobacco is a sensitive and rugged tool for targetpesticide analysis in this complex matrix. The design of thesystem enables lower detection limits for pesticides whencombined with an inert sample path and GC columnbackflushing. The high sensitivity EI Extractor Ion Source withimproved thermal characteristics delivers confident traceanalysis even in tobacco matrices, and the Triple-AxisHED-EM Detector reduces neutral noise by the doubly off-axisposition of the HED-EM. These features enabled LOQs wellbelow the GRLs issued by ACAC. Excellent linearity(R2 > 0.99) for over 92% of 162 pesticides and excellentanalysis repeatability (%RSD < 15%) for over 95% of162 pesticides in tobacco matrices were achieved. Themodified QuEChERS method using Agilent Bond ElutQuEChERS Extraction and Dispersive kits yielded excellentrecoveries from 70 to 120% for 95% of all test pesticides.

Acknowledgements

The authors thank Cynthia Cai, Chunxiao Wang, andJianguo Ji (Agilent Technologies, Inc.) for their contributions.

References

1. CORESTA Guide No. 1 - The concept and implementationof CPA guidance residue levels, July 2013. Agro-ChemicalAdvisory Committee (ACAC) of the Cooperation Centerfor Scientific Research Relative to Tobacco (CORESTA),Paris, France.

2. Anastassiades, M.; Lehotay, S. J.; Stainbaher, D.; Schenck, F. J. J. AOAC Int. 2003, 86, 412-431.

3. Lehotay, S. J.; de Kok, A.; Hiemstra, M.; Bodegraven, P.J. AOAC Int. 2005, 88, 595-614.

4. http://www.quechers.com

5. Zhao, L.; Schultz, D.; Stevens, J. Analysis of PesticideResidues in Apple Using Agilent Bond Elut QuEChERSAOAC Kits by GC/MS; Application note, AgilentTechnologies, Inc. Publication number 5990-4068EN,2012.

6. Meng, C-K. Improving Productivity and Extending ColumnLife with Backflush; Application brief, AgilentTechnologies, Inc. Publication number 5989-6018EN,2006.

7. Zhao, L.; Broske, A. D.; Mao, D.; Vickers, A. Evaluation ofthe Agilent Ultra Inert Deactivation for Active CompoundsAnalysis by GC; Application note, Agilent Technologies,Inc. Publication number 5990-7380EN, 2011.

8. Wylie, P. L.; Meng, C-K. A Method for the Trace Analysisof 175 Pesticides Using the Agilent Triple QuadrupoleGC/MS/MS; Application note, Agilent Technologies, Inc.Publication number 5990-3578EN, 2009.

9. www.ams.usda.gov/science/pdp

10. Analysis of Captan and Folpet via QuEChERS andGCMS(CI) - Brief Description. EU Reference Laboratoryfor Single Residue Methods, Fellbach, Germany.

11. Analytical Detection Limit Guidance & Laboratory Guidefor Determining Method Detection Limits, April 1996.Wisconsin Department of Natural Resources LaboratoryCertification Program, Madison, WI, USA.

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